WO2023243166A1 - Cellule destinée au traitement de brûlures et procédé de traitement de brûlures - Google Patents

Cellule destinée au traitement de brûlures et procédé de traitement de brûlures Download PDF

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WO2023243166A1
WO2023243166A1 PCT/JP2023/010225 JP2023010225W WO2023243166A1 WO 2023243166 A1 WO2023243166 A1 WO 2023243166A1 JP 2023010225 W JP2023010225 W JP 2023010225W WO 2023243166 A1 WO2023243166 A1 WO 2023243166A1
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burn
cells
placenta
burns
amniotic membrane
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PCT/JP2023/010225
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English (en)
Japanese (ja)
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大輔 小橋
淑子 吉田
素典 岡部
健一 荒井
雅彦 荒川
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国立大学法人富山大学
サクラ精機株式会社
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Publication of WO2023243166A1 publication Critical patent/WO2023243166A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/48—Reproductive organs
    • A61K35/50—Placenta; Placental stem cells; Amniotic fluid; Amnion; Amniotic stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
    • A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
    • A61L15/40—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing ingredients of undetermined constitution or reaction products thereof, e.g. plant or animal extracts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M35/00—Devices for applying media, e.g. remedies, on the human body
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents

Definitions

  • the present invention relates to cells for repairing the periphery of dermal tissue (including subcutaneous tissue) that is lost in the treatment of burns, and a method for treating burns.
  • the severity of burns is determined by area and depth. Depths are classified into degrees I to III based on the color tone of the surface. Second-degree burns are burns that reach the dermis, and third-degree burns are when all of the skin is damaged. Severe burns with degree II burns of 30% or more of the body surface area or degree III burns of 10% or more of the body surface area require intensive treatment at an emergency center. Third-degree burns in the field of emergency surgery require surgical treatment because not only the skin but also the underlying tissues, nerves, and blood vessels are damaged. Additionally, general wound dressings are not indicated due to the increased risk of infection. The area damaged by the burn is removed as soon as possible, and skin grafts are performed after the formation of granulation as a graft bed.
  • the transplanted skin has difficulty engrafting, leading to sepsis and other problems, resulting in worsening of the patient's condition and death. is often caused.
  • Non-Patent Document 1 (“Burn Treatment Guidelines [Revised 2nd Edition], 2015” published by the Japan Burn Society), reports on wound dressings are for second-degree burns, and for third-degree burns. There is no positive evidence for the use of wound dressings for degree burn wounds.
  • Wound dressings used for burns are broadly classified into foam materials, fiber materials, and colloid materials, and are used depending on their shape and exudate absorption. Both are aimed at good granulation formation.
  • Non-Patent Document 2 amniotic membrane is a tough biological membrane composed of collagen and elastic fibers, and raw amniotic membrane has been reported as a useful covering material for external injuries and burns.
  • Non-Patent Document 3 dried amniotic membrane (hyper-dry human dried amniotic membrane: hereinafter referred to as HD-AM) produced by a specific drying process has been reported (Patent Document 1, Non-Patent Document 3).
  • HD-AM dried amniotic membrane
  • the inventors created burn model animals with burn depths of degrees II to III and demonstrated that placenta-derived cells can effectively promote granulation proliferation in the dermal or subcutaneous tissue defects of burn model animals using histological and immunological studies.
  • the present invention was conceived through chemical and molecular biological confirmation.
  • the cells for treating burn injuries are cells derived from the placenta of animals including humans, which are used for treating burn injuries, and are characterized by promoting healing.
  • physiologically active substances such as inflammatory cytokines and growth factors
  • these also have active effects such as promoting the secretion of IL-6, IFN- ⁇ , IL-10, and COX2 (PGE 2 ).
  • PGE 2 COX2
  • amniotic mesenchymal stem cells may also be characterized by containing amniotic mesenchymal stem cells. According to this configuration, it has high differentiation ability and proliferation ability, and promotes wound healing in severe burns.
  • tissue regeneration for burns in the treatment of burns, and may be characterized by promoting granulation formation.
  • placenta-derived cells can be easily placed in the burn wound area.
  • the solvent may be characterized by having high viscosity. According to this configuration, placenta-derived cells can be prevented from flowing down from the burn wound area.
  • the burn may be characterized as being a second degree and/or third degree burn.
  • This configuration is particularly used for the regeneration of dermal defects or subcutaneous tissue in patients with severe burns, and promotes the secretion of physiologically active substances such as inflammatory cytokines and growth factors at the wound site of severe burns. This protects against infection by foreign substances from the outside, and also promotes wound healing in severe burns through active effects such as promoting the secretion of IL-6, IFN- ⁇ , IL-10, and COX2 (PGE 2 ). promote.
  • the cells may be characterized in that they can be used alone or together with a dressing for burns and/or a regenerated scaffold for burns.
  • the burn dressing material and/or the regenerated scaffold material for burn wounds are dried amniotic membrane obtained by drying live amniotic membrane that envelops the fetus of an animal including a human, and is dried so that it can be stored in the atmosphere.
  • the dry amniotic membrane may be characterized in that the epithelial cells, basement membrane, and connective tissue constituting living amniotic membrane are retained when the amniotic membrane is rehydrated by immersion in water or a buffer solution. According to this configuration, dried amniotic membrane can function as a cell scaffold, induce growth factors and cell migration chemokines, and promote local anti-inflammation and tissue regeneration by regulating differentiation into M2 macrophages. be.
  • the burn treatment method according to the present invention is characterized in that cells derived from the placenta of animals, including humans, are placed in the burn wound to promote healing.
  • the method when used as cells for regenerating dermal defects or subcutaneous tissues of patients with severe burns, the method promotes the secretion of physiologically active substances such as inflammatory cytokines and growth factors at the wound site of severe burns, Its inflammatory cytokines protect against infection by foreign substances from the outside, and these active actions, such as promoting the secretion of IL-6, IFN- ⁇ , IL-10, and COX2 (PGE 2 ), can prevent serious illness. Promotes wound healing in burns. In addition, safe regenerative medicine with fewer ethical problems and less immune rejection due to allogeneic transplantation is possible.
  • the placenta-derived cells may include amniotic mesenchymal stem cells. According to this configuration, it has high differentiation ability and proliferation ability, and promotes wound healing in severe burns.
  • the method may be characterized in that the placenta-derived cells are mixed with a solvent and dripped onto the burn wound site.
  • the method may be characterized by containing 100 or more of the placenta-derived cells in 50 ⁇ L of the solvent.
  • the present invention may be characterized in that 300,000 of the placenta-derived cells are contained in 50 ⁇ L of the solvent.
  • the solvent may be characterized by having high viscosity. According to this method, it is possible to prevent the solvent containing placenta-derived cells from flowing down from the burn wound site.
  • dried amniotic membrane is obtained by placing cells derived from the placenta of animals, including humans, in the burn wound area and then drying the live amniotic membrane that wraps the fetus of animals, including humans, and can be stored in the atmosphere. characterized by placing dried amnion in such a way that the epithelial cells, basement membrane, and connective tissue that make up live amniotic membrane are retained when rehydrated by immersion in water or a buffer solution. Good too.
  • dried amniotic membrane can function as a cell scaffold, induce growth factors and cell migration chemokines, and promote local anti-inflammation and tissue regeneration by regulating differentiation into M2 macrophages. be.
  • the burn may be characterized as being a second degree and/or third degree burn.
  • This method is particularly used to regenerate dermal defects or subcutaneous tissue in patients with severe burns, and promotes the secretion of physiologically active substances such as inflammatory cytokines and growth factors at the wound site of severe burns. This protects against infection by foreign substances from the outside, and also promotes wound healing in severe burns through active effects such as promoting the secretion of IL-6, IFN- ⁇ , IL-10, and COX2 (PGE 2 ). promote.
  • FIGS. 1A to 1C are diagrams illustrating the experimental mouse model and the application of HD-AM.
  • FIGS. 1A and 1B are diagrams showing the creation of a burn site in an experimental mouse.
  • FIG. 1C is a diagram showing the validity as a third degree burn experimental model.
  • Figure 2 is a diagram explaining the application of HD-AM and placenta-derived cells (Control group: schematic diagram of mice without HD-AM and placenta-derived cells, Cell group: mice using placenta-derived cells). (Schematic diagram of HD-AM group: Schematic diagram of mice using HD-AM, HD-AM/Cell group: Schematic diagram of mice using HD-AM and placenta-derived cells).
  • 3A and 3B are schematic illustrations of a method for measuring granulation tissue thickness.
  • FIG. 1A and 1C are diagrams illustrating the experimental mouse model and the application of HD-AM.
  • FIGS. 1A and 1B are diagrams showing the creation of a burn site in an experimental mouse.
  • FIG. 3A is a diagram illustrating temporal observations of tissues stained by Azan staining, which selectively stains connective tissue, after a burn injury.
  • FIG. 3B is a schematic diagram showing a method for measuring the thickness of granulation formed during treatment after a burn injury.
  • Figure 4 is a table showing the primers used in quantitative reverse transcription polymerase chain reaction (q RT-PCR).
  • Figures 5A and 5B are graphs comparing photomicrographs of granulation formation over time and average granulation tissue thickness during post-injury treatment.
  • FIG. 5A is a photomicrograph of the Control group, Cell group, HD-AM group, and HD-AM/Cell group on days 1, 4, and 7 after treatment (POD1, POD4, and POD7).
  • FIGS. 6A to 6C are diagrams illustrating blood vessel formation within granulation tissue.
  • FIG. 6A is a photograph showing the state of POD4 angiogenesis.
  • FIG. 6B is a photograph showing the state of angiogenesis in POD7.
  • FIG. 6C is a graph comparing the measurement sites of blood vessels formed within the granulation tissue and the average of the measurement values.
  • FIGS. 7A to 7D are graphs comparing inflammatory and anti-inflammatory cytokines using quantitative RT-PCR.
  • FIG. 7A is a graph showing mRNA expression of cytokines involved in inflammation.
  • FIG. 7A is a graph showing mRNA expression of cytokines involved in inflammation.
  • FIG. 7B is a graph and a photograph showing the expression of mRNA for the inflammatory cytokine IL-6 and the distribution of IL-6.
  • FIG. 7C is a graph and a photograph showing the expression of mRNA of a Type II macrophage marker (CD163) involved in anti-inflammation and the distribution of Type II macrophages.
  • FIG. 7D is a graph and a photograph showing the expression of mRNA for the inflammatory cytokine IL-10 and the distribution of IL-10.
  • FIG. 8 is a diagram showing a drying apparatus for producing dried amniotic membrane (HD-AM).
  • Placenta-derived cells are amniotic mesenchymal cells and amniotic mesenchymal stem cells in the following experiments, but are not limited to these.
  • Amnion is an extraembryonic tissue composed of ectoderm-derived epithelial cells and mesoderm-derived mesenchymal cells, and contains a group of cells that have characteristics as pluripotent stem cells. Since amniotic membrane is discarded as excrement after childbirth, there are few ethical problems when using it as a biomaterial. In addition, amniotic membrane has special immunological properties and has low immunogenicity, so that the immune rejection reaction caused by allotransplantation is relatively mild.
  • Placenta-derived cells are collected from the amniotic membrane of mammals including humans, but amniotic membrane is composed of epithelial cells and mesenchymal cells, and in order to collect a cell population of mesenchymal cells from amniotic membrane, amniotic membrane
  • the epithelial cells may be removed from the cell and a separation operation may be performed.
  • Collection of a cell population of mesenchymal cells can be performed, for example, according to the method described in JP-A No. 2003-231639.
  • human amniotic membrane can be collected, for example, from a pregnant woman with informed consent by caesarean section.
  • the collected amniotic mesenchymal cell population contains a mixture of cells with various proliferative abilities, lifespans, and properties. Therefore, when maintaining a mesenchymal cell population under general culture conditions, some of the cells included in the mesenchymal cell population adhere and begin to proliferate at the beginning of the culture. Since epithelial-like cells occupy most of the culture surface, amniotic mesenchymal stem cells, which begin to proliferate later than the epithelial-like cells, cannot proliferate and are expelled, making it impossible to isolate them.
  • the cell population of amniotic mesenchymal stem cells includes cells with a spindle-like morphology and has a high proliferation ability, preferably capable of cell division (population doublings) 50 times or more.
  • placenta-derived cells When placenta-derived cells are used for the treatment of burn wounds, for example, 100 or more placenta-derived cells are contained per 50 ⁇ L of solvent, preferably 300,000 placenta-derived cells are contained per 50 ⁇ L of solvent, and placenta-derived cells are added to the burn wound area. Placenta-derived cells can be placed at the burn site by dropping a cell-containing solution. In addition, when dropping a solution containing placenta-derived cells onto a burn wound, use a bandage, etc., over the solution containing placenta-derived cells to prevent the solution from flowing. It is recommended to apply a wound dressing to the burn wound.
  • HD-AM dried amniotic membrane
  • placenta-derived cells may be used alone or in combination with dried amniotic membrane (HD-AM), which will be described later.
  • the dried amniotic membrane produced by a specific drying process is, for example, the dried amniotic membrane described in Patent Document 1.
  • the raw amniotic membrane placed in the processing tank is continuously heated by an infrared heater installed in the processing tank, and the inside of the processing tank is depressurized.
  • the raw amniotic membrane is irradiated with microwaves from a microwave generator installed in the processing tank to dry the amniotic membrane while adding energy to the water molecules present in the amniotic membrane.
  • dried amniotic membrane produced by repeating this process multiple times, the amniotic membrane cells themselves are inactivated, but their cell and tissue structure is maintained. Specifically, dried amniotic membrane (HD-AM) is dried so that it can be stored in the atmosphere, and amniotic membrane that has been rehydrated by immersing it in water or a buffer solution contains epithelial cells that constitute live amniotic membrane, The basement membrane and connective tissue are retained.
  • experiments were conducted to form good granulation tissue using placenta-derived cells alone or in combination with HD-AM.
  • HD-AM as a dressing and scaffolding material in severe burn wounds, inflammatory effect cells including Type I macrophages appear, and inflammatory cytokines are transiently increased to protect against infection and promote angiogenesis.
  • anti-inflammatory cytokines increase, promoting active effects such as high-quality granulation formation.
  • HD-AM can be used as a dressing material and a scaffold material by molding it into an appropriate shape with scissors, etc., depending on the wound site, such as a burn or severe burn where general wound dressings cannot be used. Furthermore, it is preferable to create drainage holes in the coated HD-AM for draining exudate and the like from the body.
  • FIGs 1A to 1C and 2 are diagrams illustrating the experimental mouse model and the application of HD-AM. After the mouse is anesthetized and placed in a prone position, its limbs are immobilized. Next, the skin on the back is shaved and coated with hair removal cream. Next, using a tube on the back (FIG. 1A), a 10 mm area was exposed to hot water at 90 degrees for 10 seconds to form a third degree burn wound with a diameter of 10 mm in the center of the back (FIG. 1B).
  • Figure 1C shows that in a third-degree burn experimental model, after being treated with boiling water at 90 degrees Celsius for 10 seconds, the exposed skin immediately turns pale, and on the seventh day, the entire skin layer becomes necrotic and falls off. Based on these results, this experimental model was judged to be appropriate as a third-degree burn model, and was used as a third-degree burn model to examine the effects of burn treatment such as granulation formation.
  • the experimental animals were handled in accordance with the guidelines of the National Institutes of Health, and permission was obtained from the University of Toyama Animal Care and Use Committee. Furthermore, the experiment was conducted in accordance with the guidelines of the Toyama University Animal Experiment Committee.
  • FIG. 2 shows an explanatory diagram of the use of HD-AM and/or placenta-derived cells in burn treatment.
  • a case is explained in which amniotic mesenchymal stem cells are used as placenta-derived cells.
  • Cell group a group using placenta-derived cells
  • HD-AM group a group using HD-AM
  • HD-AM/Cell group a group using HD-AM and placenta-derived cells
  • control group Four groups were created, including a group in which neither was used (control group).
  • Each group consisted of 6 mice and was evaluated on postoperative day 1 (POD 1), postoperative day 4 (POD 4), and postoperative day 7 (POD 7), for a total of 72 mice.
  • POD 1 postoperative day 1
  • POD 4 postoperative day 4
  • POD 7 postoperative day 7
  • the burn wound (wall of exposed bowl) in the mouse model was covered with a polyurethane foam dressing (Tegaderm® Diamond transparent film®, 3M Deutschiand GmbH Health Care Business, Germany) and stainless steel. Covered with mesh (0.06mm ⁇ , 150mesh).
  • HD-AM was placed on a wall of exposed bowl in a mouse model, and a polyurethane foam dressing (Tegaderm® Diamond transparent film®, 3M Deutschiand GmbH Health Care Business, Germany) and then covered with stainless steel mesh (0.06 mm ⁇ , 150 mesh).
  • HD-AM/Cell group 50 ⁇ L of a solution containing 300,000 placenta-derived cells was dropped onto the wall of exposed bowl of a mouse model, HD-AM was placed on top of the wall of exposed bowl, and then polyurethane foam was placed on top of it.
  • placenta-derived cells may be included in a cell culture medium or a jelly-like medium so that the placenta-derived cells do not flow away from a burn wound.
  • agar can be used as the jelly-like medium for cell culture. Jelly-like refers to a highly viscous fluid that has not lost its fluidity. Viscosity may be added to saline or phosphate-buffered saline (PBS).
  • stainless steel mesh is used to cover the HD-AM, wound dressing, etc. so that they do not dislodge due to mouse behavior.
  • FIGS. 3A and 3B show schematic diagrams of a method for measuring the thickness of granulation tissue.
  • Figure 3A is a diagram showing the temporal observation of tissues stained by the Azan staining method, which selectively stains connective tissue after a burn injury. 2, and the layer below the collagen layer was designated as tissue 3.
  • FIG. 3B is a schematic diagram showing a site where the thickness of granulation tissue formed in a wound area is measured during treatment after a burn injury.
  • tissues 1, 2, and 3 were distinguished, and granulation tissue newly constructed in tissue 3 was measured.
  • the schematic diagram of FIG. 3B shows that tissue 3 is located in the upper layer of adipose tissue.
  • Azan staining distinguished collagen fibers from fibrin and facilitated observation of newly constructed granulation tissue.
  • the thickness of granulation tissue containing collagen fibers was measured using the Olympus® CellSens® imaging program (version 1.7; Olympus, Tokyo, Japan).
  • POD4 On the fourth day after treatment (POD4), a layer of collagen membrane was observed between the subcutaneous tissue and muscle layer of the surrounding normal tissue, regardless of whether HD-AM was attached or not. Since the movement of infiltrating cells and granulation formation were observed across this membrane, each region was determined as shown in FIG. 3A. As shown in FIG.
  • the measurement sites were three points: the epithelial defect stump a, the center b of the epithelial defect stump, and the midpoint c between a and b.
  • the thickness of the granulation tissue formed in the tissue 3 at these three points a, b, and c was measured, and the average value was taken as the thickness of the granulation tissue.
  • FIG. 4 shows the sequences of the primers used in quantitative reverse transcription polymerase chain reaction (q RT-PCR).
  • q RT-PCR quantitative reverse transcription polymerase chain reaction
  • RNA was treated with deoxyribonuclease I (DNase I, Sigma-Aldrich, Inc., Tokyo, Japan) for 15 minutes at room temperature.
  • cDNA was synthesized using 500 ng of DNase I-treated RNA using the Rever Tra Ace qPCR RT Kit (Toyobo Co., Ltd., Osaka, Japan).
  • Gene expression was measured using the Mx3000P quantitative polymerase chain reaction (qPCR) system (Stratagene; Agilent Technologies Japan Ltd. Japan) and real-time RT-PCR using the Brilliant SYBR Green QRT-PCR Mix (Stratagene; Agilent Technologies Japan Ltd. Japan). Quantitated by PCR analysis.
  • qPCR quantitative polymerase chain reaction
  • mRNA was extracted from each group of 6 mice on POD1, POD4, and POD7, and mRNA was extracted from each group of 6 mice to detect inducible nitric oxide synthase (iNOS) produced by Type I macrophages, CD163 which is an indicator of Type II macrophages, inflammatory cytokines IL-6, and IFN. - ⁇ and COX-2, which induces PGE 2 involved in angiogenesis, were measured in a single manner.
  • iNOS inducible nitric oxide synthase
  • FIG. 5A is representative micrographs of the Control group, Cell group, HD-AM group, and HD-AM/Cell group on days 1, 4, and 7 after treatment (POD1, POD4, and POD7).
  • the thickness of granulation tissue in the wound area of each group is indicated by black arrows.
  • FIG. 6A shows the state of POD4 angiogenesis in each group other than the Control group.
  • CD31-positive cells and ⁇ -SMA-positive cells were observed within the granulation tissue.
  • images were observed in which CD31-positive cells constituted the vascular lumen and ⁇ -SMA-positive cells surrounded them.
  • HD-AM group CD31-positive cells were present but no vascular lumen was observed.
  • FIG. 6B shows the state of angiogenesis in POD7 in each group other than the Control group.
  • the HD-AM group CD31-positive cells and ⁇ -SMA-positive cells were observed to aggregate, forming blood vessels within the granulation.
  • the number of these cells decreased and they were observed running as complete blood vessels. Blood vessel formation was observed in deeper layers in the Cell group and HD-AM/Cell group compared to the HD-AM group.
  • Figure 6C shows a comparison of angiogenesis within the granulation tissue.
  • the photo on the left shows an example of granulation tissue magnified using a 20x objective lens, and the inner circumference of a blood vessel formed by CD31-positive cells present in one field of view is measured in each group at POD4 and POD7. It was measured. The measured results are shown in the graph on the right. Looking at the graph of POD4, in POD4, the length of blood vessels in the Control group is extremely short and close to 0, but the length of blood vessels is significantly shorter in the Cell group, HD-AM group, and HD-AM/Cell group. increased.
  • the length is approximately 1500 ⁇ m in the Cell group, approximately 1100 ⁇ m in the HD-AM group, and approximately 2500 ⁇ m in the HD-AM/Cell group.
  • the Cell group showed a significant increase in the length of blood vessels at POD7, with a length of approximately 5700 ⁇ m.
  • the number of blood vessels was small, blood vessels were observed running in a wide area (from the superficial layer to the deep layer).
  • Figures 7A to 7D show the expression of mRNA for markers and cytokines of cells involved in inflammation.
  • the expression level of iNOS (Type I macrophage) tends to increase in the POD4 cell group, HD-AM group, and HD-AM/cell group, with a tendency for the HD-AM/Cell group > Cell group > HD-AM group. It was seen. At POD7, the expression of iNOS (Type I macrophages) tended to decrease in all groups.
  • COX-2 The expression level of COX-2 (PGE 2 ) tended to increase in all groups at POD4. At POD7, the expression level tended to decrease in all groups other than the control group. In this way, COX2, which is present upstream in the production process of PGE 2 that causes angiogenesis, increases at POD4 and tends to decrease at POD7 in all groups, supporting the results of immunohistochemistry (CD31 and ⁇ SMA). there were. Regarding POD4, the expression level of the inflammatory cytokine IFN- ⁇ tended to be higher in the cell group and HD-AM/cell group than in the control group and HD-AM group. At POD7, expression tended to decrease in all groups.
  • FIG. 7B shows the mRNA expression of the inflammatory cytokine IL-6 and the distribution of IL-6.
  • the expression level of IL-6 mRNA increased on POD4 compared to POD1, and the expression tended to decrease on POD7.
  • the results of immunohistochemical staining (POD4, cell group) showed that IL-6 positive cells were diffusely present within the granulation tissue.
  • FIG. 7C shows the expression pattern of Type II macrophage marker-related mRNA (CD163) and the distribution of CD163-positive cells.
  • Type II M ⁇ Although there was almost no expression in POD1, the expression tended to increase in all groups in POD4. At POD7, expression tended to increase in the HD-AM group.
  • the immunohistochemically stained granulation tissue POD7, HD-AM/cell group
  • CD163-positive cells were clearly observed in the newly formed granulation tissue at POD7.
  • FIG. 7D shows the mRNA expression of the inflammatory cytokine IL-10 and the distribution of IL-10.
  • the expression level of IL-10 mRNA was significantly higher in the HD-AM/cell group than in the control group and HD-AM group.
  • HD-AM Dried amniotic membrane
  • a depressurization operation in which the raw amniotic membrane placed on the rotary table 12 in the processing tank 10 is continuously heated by a far-infrared heater 14 provided in the processing tank 10 to bring the inside of the processing tank 10 into a depressurized state;
  • the raw amniotic membrane is also irradiated with microwaves from the microwave irradiation device 30 installed in the treatment tank to remove the water molecules present in the amniotic membrane. Drying is performed while applying energy to the By repeating this process multiple times, the amniotic membrane that is dried while retaining its cell and tissue structure has improved storage stability and is easier to handle.
  • Cells derived from the placenta of animals, including humans, are used as materials for regenerative medicine, especially in the treatment of severe burns.
  • dried amniotic membrane as a medical device in combination with placenta-derived cells, it is possible to cover the dermal defect or subcutaneous tissue of severe burn patients where placenta-derived cells have been placed, and to perform skin grafting.
  • tissue regeneration favorable granulation formation accompanied by angiogenesis
  • skin grafting which is essential for third-degree burns, can be achieved at an early stage.
  • it to prevent infection in burn wounds, it is possible to increase patient survival rates and treatment effects (no worries about keloids).

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  • Bioinformatics & Cheminformatics (AREA)
  • Oral & Maxillofacial Surgery (AREA)
  • Transplantation (AREA)
  • Rheumatology (AREA)
  • Pain & Pain Management (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Pregnancy & Childbirth (AREA)
  • Reproductive Health (AREA)
  • Biotechnology (AREA)
  • Immunology (AREA)
  • Virology (AREA)
  • Anesthesiology (AREA)
  • Materials Engineering (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Media Introduction/Drainage Providing Device (AREA)
  • Materials For Medical Uses (AREA)

Abstract

La présente invention aborde le problème de promotion de manière efficace de la granulation dans une altération du derme ou une altération du tissu sous-cutané provoquée par une brûlure grave. Pour résoudre ce problème, l'invention concerne une cellule à utiliser pour traiter une brûlure, ladite cellule étant dérivée du placenta d'un animal, notamment des êtres humains, et favorisant la cicatrisation.
PCT/JP2023/010225 2022-06-17 2023-03-16 Cellule destinée au traitement de brûlures et procédé de traitement de brûlures WO2023243166A1 (fr)

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JP2022097967A JP2023184057A (ja) 2022-06-17 2022-06-17 熱傷治療用の細胞及び熱傷治療方法
JP2022-097967 2022-06-17

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007046775A1 (fr) * 2005-10-21 2007-04-26 Cellresearch Corporation Pte Ltd Isolation et culture de cellules souches/génitrices à partir de la membrane amniotique de cordon ombilical et utilisations des cellules différenciées obtenues ainsi
JP2021020868A (ja) * 2019-07-26 2021-02-18 国立大学法人富山大学 熱傷用修復材

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007046775A1 (fr) * 2005-10-21 2007-04-26 Cellresearch Corporation Pte Ltd Isolation et culture de cellules souches/génitrices à partir de la membrane amniotique de cordon ombilical et utilisations des cellules différenciées obtenues ainsi
JP2021020868A (ja) * 2019-07-26 2021-02-18 国立大学法人富山大学 熱傷用修復材

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
DUA HARMINDER S.; TING DARREN SHU JENG; AL SAADI AHMED; SAID DALIA G.: "Chemical eye injury: pathophysiology, assessment and management", EYE, NATURE PUBLISHING GROUP, GB, vol. 34, no. 11, 22 June 2020 (2020-06-22), GB , pages 2001 - 2019, XP037274403, ISSN: 0950-222X, DOI: 10.1038/s41433-020-1026-6 *
KITALA DIANA; ŁABUś WOJCIECH; KLAMA-BARYłA AGNIESZKA; KRAUT MAłGORZATA; MAJ MARIUSZ; SZAPSKI MICHAł: "Application of Amniotic Stem Cells on an Acellular Dermal Matrix Scaffold in a Burned Patient: A Case Report", TRANSPLANTATION PROCEEDINGS, ELSEVIER INC., ORLANDO, FL; US, vol. 52, no. 8, 19 May 2020 (2020-05-19), ORLANDO, FL; US , pages 2563 - 2569, XP086260454, ISSN: 0041-1345, DOI: 10.1016/j.transproceed.2020.01.110 *
LI JING-YUAN, REN KANG-KANG, ZHANG WEN-JIE, XIAO LING, WU HAN-YOU, LIU QIAN-YU, DING TING, ZHANG XIANG-CHENG, NIE WEN-JIA, KE YU, : "Human amniotic mesenchymal stem cells and their paracrine factors promote wound healing by inhibiting heat stress-induced skin cell apoptosis and enhancing their proliferation through activating PI3K/AKT signaling pathway", STEM CELL RESEARCH & THERAPY, vol. 10, no. 247, 9 August 2019 (2019-08-09), pages 1 - 17, XP055817551, DOI: 10.1186/s13287-019-1366-y *
ZENG WEI, LI YANWEI, ZENG GUANGWEI, YANG BO, ZHU YU: "Transplantation with cultured stem cells derived from the human amniotic membrane for corneal alkali burns: an experimental study", ANNALS OF CLINICAL AND LABORATORY SCIENCEUNITED STATESNOV 2021, vol. 44, no. 1, 1 January 2014 (2014-01-01), pages 73 - 81, XP093118221, ISSN: 1550-8080 *

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